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Thawing Strains
When you need to get a yeast strain out of the -80C, here is what you do. You'll need YPD plates, 200ul tips, latex gloves, toothpicks, Kimwipes, 95% EtOH, 2 days' use of the 30C Incubator. Day One 1. Find out the details about your strain, from the lab yeast strain database files. These files are available from our lab FTP server, see Getting FTP Files if you don't have them or need a newer version. There are excel files for the a, alpha, and heterozygous diploid deletion collections, and for strains created by certain people and people who worked with them, such as Mitsu or Kristan. You can open these files with Microsoft Office, or if you don't have that, download LibreOffice, which is free and available for Mac, Windows, and Linux among others. Use the Find feature, typically Ctrl-F or Edit>Find, to search for your strain. This will let you know in some cases where it is in the freezer, as well as additional details about your strain. If you only know your gene's name, for example STE5, and the Excel file you want to use (e.g. the Het Dip deletion collection) has only the systematic names, for example YDR103W, you'll need to go to SGD and type the gene name into the search bar to find the systematic name, first. 2. Using a Sharpie, draw pie slices on the bottom of your YPD plate to make up to 6 slices, one for each strain you will put on that plate, like slicing a pizza. When you're starting, 4 slices is probably as crowded as you want to try. ONLY STRAINS OF THE SAME MATING TYPE SHOULD GO ON A PLATE TOGETHER. 3. On the outside bottom of each pie slice, in fine-point Sharpie, write the strain that will go in that slice. Somewhere on the plate add your name or initials, and the date. 4. Wearing latex gloves, take your YPD plates, 200ul tips, and list of strains to the -80C Chest Freezer in the equipment corridor, across from the Bioscreen. There is a sticker on the front of the chest freezer that lists which strains and which strain collections are in each rack within the freezer. It's cryptic and not currently always up to date; when in doubt about which rack you need, poke around or ask someone. If the strain starts with two letters (e.g. BR2200), those are the initials of the person who made the strain, and if the initials ring a bell that's a good person to ask first if you can't find the strain. The RLS dissectors also usually know where everything is. 6. Remove the rack you need from the freezer and set it on top of the remaining racks. Remove the wire that holds the boxes in the rack by lifting it from the top. The boxes are labeled in sharpie; remove the one with your strain. Take out the 96-well plate or 1.5 ml Eppendorf tube with your strain in it. For tubes, open, stab your tip into the frozen strain until you see anything at all on the end, and dab it on the outer area of the appropriate pie slice on your plate, where you should see a tiny drop. For plates: wipe down the foil with 95% EtOH and a Kimwipe, both should be on the sink to the left of the freezer. Stab through the foil of your well (the letters and numbers for the wells are along the edges of the plates in raised type beneath the foil), dab on plate, use a tiny square of foil to reseal the hole. Put away your box and rack if you're done; don't forget to replace the wire holding the boxes into the rack. Repeat for all strains. 7. Return to your bench. Using a toothpick, make a 4-phase streak in your pie slice. Here's how: take out a sterile toothpick carefully by one end. Sweep your toothpick gently through the dab on the plate in an S motion, staying inside the pie slice. Flip the toothpick over, and sweep the new side through the end of the previous S, continuing inside the pie slice. Take a second toothpick and continue front and back, for four connected S's inside your slice. Each new side/toothpick should pick up a bit of the last one's trail, so that you're diluting out your yeast as you go to be sure to have single colony dots later. 8. Put plates in the 30C Incubator overnight. Day Two 9. Using a toothpick, the next day pick a single colony from your streak for each of your strains, and re-streak it. Do this by taking a dab of that colony and dabbing it onto the pie-slice of a new, freshly labeled YPD plate, and then sweeping through that dab to make a new 4-phase streak for each strain, as before. When these plates grow up, you have your thawed strain and can proceed. N.B.: If at step 9 you look at your overnight plates from the freezer and there are clearly two different sizes of colonies visible on a pie slice, that strain may actually be two strains mixed together, and you can't be sure which is the correct one. If you're worried that this may be the case, stop and talk to someone in lab about how to proceed for that particular strain from there.